平肝熄风法调节α-synuclein基因表达对多巴胺能神经元的保护作用的研究

Parkinson's disease (PD) is a degenerative disorder of the central nervous system, characterized by the progressive loss of dopaminergic neurons in the substantia nigra, that mainly affects elderly people. Neuroprotective therapy for PD is radical treatment. The α-synuclein gene is causatively related to PD. Liver-Yang Hyperactivity Syndrome play a critical role in the TCM pathogenesis of PD. Therapeutical principle of sedate the liver and extinguish wind is effective in treating the patients with PD via protection of neurons. However, the underlying mechanism remains unknown. This project take α-synuclein gene as the breakthrough point to research neuroprotective effect and α-synuclein inhibitive effect of Zhenganxifeng Decoction in vitro and in vivo by modified cerebrospinal fluid pharmacology, real-time RT-PCR, Western blot, RNA interference, double-label immunofluorescence assays, and further explore the mechanism by which the neuroprotective effect of it are associated with reduction of the mitochondrial pathway of apoptosis mediated by α-synuclein. This project aims at clarifying action mechanism and therapeutic target of therapeutical principle of sedate the liver and extinguish wind therapy for PD, and offering the experimental platform for research on Chinese compound prescription herbs of therapeutical principle of sedate the liver and extinguish wind, and offer fresh ideas, new thinking, and new methods for re-development of TCM prescription.

帕金森病（Parkinson's disease, PD）是中老年人群中常见的神经系统退行性疾病，主要病理变化是黑质多巴胺神经元损伤，神经保护治疗是PD的根本治疗。α-synuclein基因与PD密切相关。肝阳上亢是PD的病机关键，平肝熄风治法的神经保护和防治PD的作用显著，但其作用机制尚未阐明。本项目以α-synuclein基因为切入点，应用改良脑脊液药理学、实时定量RT-PCR、Western blot、RNA干扰、荧光双标染色等实验技术，分别在PD细胞模型和动物模型上，研究平肝熄风治法代表方镇肝熄风汤的神经保护作用和对α-synuclein基因表达的影响，并进一步探讨其通过抑制α-synuclein介导的线粒体凋亡途径实现神经保护作用。以期阐明平肝熄风法治疗PD的作用机制和靶点，为平肝熄风治法方药研究提供可靠的实验研究平台，为我国中药复方二次开发提供了新观点、新思路和新方法。

本项目研究按计划完成了相关内容。我们研究发现，帕金森病（Parkinson disease, PD）模型小鼠爬杆实验评分值和自主活动计数实验移动格子数较正常组显著减少，镇肝熄风汤呈剂量依赖性的增加PD小鼠爬杆评分值和自主活动计数实验移动格子数。PD模型小鼠小脑组织酪氨酸羟化酶（tyrosine hydroxylase, TH）和14-3-3 mRNA和蛋白表达较正常组显著下调，而α-突触核蛋白（α-synuclein, α-syn）mRNA和蛋白表达较正常组显著上调，镇肝熄风汤具有上调PD模型小鼠小脑组织TH和14-3-3 mRNA和蛋白表达，而下调α-Syn mRNA和蛋白表达的作用。通过流式细胞仪FITC-Annexin V染色、caspase-3和caspase-9活性等指标观察显示，PD模型组小鼠小脑神经元凋亡较正常组显著增多，而镇肝熄风汤呈剂量依赖性的减少PD小鼠小脑神经元凋亡。PD模型组小鼠小脑组织Bax mRNA和蛋白表达较正常组显著上调，细胞色素c（cytochrome c，Cyt-C）蛋白表达上调，而Bcl-2和Bcl-xL mRNA和蛋白表达明显下调，镇肝熄风汤具有抑制PD模型小鼠小脑组织Bax mRNA和蛋白表达，下调Cyt-C蛋白表达，而上调Bcl-2和Bcl-xL mRNA和蛋白表达的作用。我们进一步体外研究发现，MTT法检测结果显示， MPP+处理诱导PC12细胞活力显著下降，镇肝熄风汤含药脑脊液（CSF-ZGXF）可呈浓度依赖性的升高MPP+损伤的PC12细胞活力。DNA片断法和caspase-3活性分析结果表明，MPP+处理诱导了PC12细胞凋亡，而CSF-ZGXF预处理呈剂量依赖性的抑制了MPP+诱导的PC12细胞凋亡。CSF-ZGXF预处理抑制了MPP+诱导的PC12细胞Cyt-C释放。MPP+处理诱导了PC12细胞Bcl-2 mRNA和蛋白表达下调及Bad mRNA和蛋白表达上调，而CSF-ZGXF处理可呈浓度依赖性的增加了MPP+处理PC12细胞的Bcl-2 mRNA和蛋白表达，而减少了Bad mRNA和蛋白表达。上述研究结果部分阐明了镇肝熄风汤防治PD的作用机制，为平肝熄风法防治PD的临床应用提供了药理学依据。本项目研究结果在中国中文核心期刊源发表论文2篇，获授权实用新型专利1项。